This invention is in the field of mass spectrometry and more specifically relates to the separation, collection and quantification of components of mixtures using a magnetic analyzer coupled with a collection array. The method is of particular application to separation of biologically-active components from mixtures from biological samples such as natural products, peptides, polynucleotides, proteins and polysaccharides. Biological samples include various types of samples (gas, liquid, or solid) from various biological environments, e.g., various human or veterinary medical samples (blood, urine, etc.), samples of bacteria, fungi or other microorganisms; water, soil of air samples, etc.)(gas, liquid, or solid.) The method is also useful for the separation of synthetic organic components from complex mixtures, such as combinatorial libraries.
Mass spectrometers use various combinations of electric and magnetic fields to achieve spatial or temporal separation of ions in the rarefied gas phase (1). In addition to the analytical utility of mass spectrometry, spatial separation of ions by mass spectrometric methods has been considered previously in conjunction with preparative separation of selected components of mixtures. For example, mass spectrometers were used in the Manhattan Project for the separation of the 235U isotope from the much more abundant 238U isotope (2-4). The mixture of isotopes was atomized and ionized in an efficient ion source, separated by a homogeneous magnetic field and landed on a collector. This was done under destructive conditions that excluded survival of molecular species because of the ionizing conditions and the high kinetic energies with which the ions impinged on the collector (3,4). Recently several attempts have been made to soft land gas phase ions following mass separation by a mass spectrometer (5-17). The term xe2x80x9csoft landingxe2x80x9d usually refers to and is used herein to refer to, non-destructive capture of a gas-phase ion on a target, such that it can be retrieved from the vacuum system of the mass spectrometer and identified or otherwise analyzed or used. Soft landing is not always required for identification of a mixture component, but is essential for efficient, high-yield preparative separation of mixture components for further analysis, functional assays or use. Mass separation or separation by mass refers to separation of ions possessing different mass to charge ratios (m/z). When the ions generated are singly charged, m/z values can be replaced by and referred to as masses.
Examples of soft landing of ions include a polypropylene glycol oligomer (5), chlorobenzyl ions (6), sulfonium ions (7), a mixture of multiply charged DNA fragments (8), CO2 (9) and inorganic metal clusters (10-12). The targets used for soft landing of ions include metal surfaces (5, 7), inert gas matrices (9-12), nitrocellulose membranes (8) and self-assembled monolayers (6,13,14). Mass separation in these examples was achieved by mass spectrometers including quadrupole mass filters (5,6,9,13-16), a sector instrument (7), and an ion-cyclotron resonance instrument (8).
These references all exemplify single-channel ion collection and isolation. In single-channel collection, ions are mass selected by tuning a mass spectrometer to a selected mass and collecting only ions having the selected mass. Collection of a second ion requires retuning of the mass spectrometer to select the mass of the second ion and collection of the second ion. The application of single-channel ion collection to component separation can be prohibitively time consuming for practical application, particularly when separation of multiple components of complex mixtures is desired.
Thus, prior art efforts to achieve separation can be characterized as single-channel isolation of mass-selected ions in slightly modified commercial or existing mass spectrometers. The yields of soft-landed ions using such methods have not been quantified. Feng et al. (8) estimate capture of attomole amounts of DNA, while Geiger et al. (7) report reanalysis of a soft-landed sample collected xe2x80x9covernightxe2x80x9d by fast-atom bombardment mass spectrometry, which typically requires at least picomole amounts of sample. Thus, while the prior art suggests that soft landing of a variety of mass-selected gas phase ions is possible, the implementation of component separation using soft landing of ions lacks practical implementation.
There remains a significant need in the art for improved mass spectrometer-based methods for separation of components of mixtures that are sufficiently efficient and high yield for practical application.
The present invention provides an instrument and methods for the preparative separation of components of mixtures using mass spectrometric methods. Nondestructive ionization methods are employed to generate ionized components of a mixture, the ionized components are spatially separated by mass and the mass-separated ion components are trapped. The ion source and mass spectrometric techniques employed allow the generation of large ion currents of ion components, on the order of nanoamps, which facilitate rapid accumulation of nanomole quantities of mass-separated components in relatively short times (minutes to hours).
The method of this invention can, for example, provide several nanomoles of a compound of interest for 10 h of collection of ions generated at 10 nA ion current by electrospray ionization. The amount of time needed to accumulate a nanomole of material depends on the abundance of the component in a mixture (e.g., its molarity) and the ionization efficiency of the component (e.g., its electrospray ionization efficiency). In order to obtain 10 picomol of a biological sample for biological testing, the collection time would be about 100s for an ion generated at about 10 nA ion current and about 1000s for an ion generated at about 1 nA ion current. Products can be collected at a rate of about 10 picomole/h or more and, preferably, at a rate of about 50 picomole/h or more. Note that the collection time for multiple components from the same sample is significantly decreased in the method of this invention because multiple components can be mass dispersed and collected simultaneously. A plurality of ion components from a mixture can typically be collected in less time than has been needed in prior art methods to collect a single ion component.
Typical samples for preparative electrospray mass spectrometry are in the range of about 5xc3x9710xe2x88x925 to about 1xc3x9710xe2x88x924 M/component. Samples for preparative electrospray mass spectrometry are typically solutions in volatile water-miscible solvents, such as water, volatile alcohols (methanol, ethanol, etc.) acetonitrile, nitromethane, tetrahydrofuran, and volatile organic acids (formic acid, acetic acid, propionic acid, etc.).
Mixtures are subject to non-destructive ionization, preferably using atmospheric pressure ionization techniques, such as electrospray ionization or atmospheric pressure chemical ionization techniques, to generate ionized components of the mixture. These ionized components are transmitted into a high vacuum region, where they are accelerated to high kinetic energy (on the order of kilo electron volts, keV). The ions generated in the ion source at higher pressures (about 1 Torr) are transported employing ion lensing and ion guiding to the high vacuum region (about 10xe2x88x926 Torr). Accelerated ions are energy selected in an electrostatic analyzer and passed into a magnetic analyzer where they are dispersed by mass. The mass-dispersed ions are decelerated to low kinetic energy (about 15 eV or less) and trapped on a collector array where the location of trapping on the array depends on the mass of the trapped ion. Ions can, for example, be collected according to mass into an array of collector compartments or bins. Bins or compartments are sized, spaced and arrayed along the length of the collector each to receive ionic species of different m/z or to receive ionic species having m/z of a selected range.
Mass separation occurs simultaneously for all ion components providing a 100% duty cycle. Trapping of all mass-separated ions also occurs simultaneously allowing for multiplex separation which facilitates analysis or biological testing of separated components. Ion generation, mass-separation and ion-trapping of all components are continuous for a given sample and do not require mass scanning or mechanical movement of the collector array to achieve separation of components. The instrumentation and method of this invention are particularly well suited to separation and analysis of complex mixtures, for example, complex samples from biological sources. The instrumentation and method of this invention can, for example, be employed in the separation and screening of natural product mixtures (e.g., including, peptides, proteins or polynucleotides) as well as combinatorial libraries (e.g., including various organic species or biological molecules (including peptides, proteins, and polynucleotides) for the identification of components with desirable biological or chemical properties or reactivity. The method is efficient and high yield rapidly providing sufficient amounts of separated materials (picomole quantities or greater) for functional, chemical or other types of analysis. In contrast to other methods for separating compounds by molecular weight (e.g. capillary electrophoresis, diffusion methods, etc.), the current invention can provide separation of relatively small molecules according to their mass/charge ratios at a resolution approaching 1 Dalton mass difference.
The use of high velocity ions reduces space-charge effects allowing the generation of high ion currents in the instrument. The high kinetic energy ions can nevertheless be soft-landed onto a collection surface at low velocity and kinetic energy by use of a deceleration lens to maximize non-destructive capture of mixture components. The collection surface can be, for example, a conducting metal, a polymer, or more generally, any non-volatile matrix. When it is desired to capture mixture components without substantial structural change, the collection surface preferably does not react with the ionic species that are landed. The collection surface may however, function to neutralize the charge of the ionic species landed. Alternative, it may be desirable to land the ionic species on a collection surface that is reactive to generate a desired reaction product of the landed species. Furthermore, it may be desirable to land the ionic species at a controlled velocity and kinetic energy to generate fragments or to enhance reactivity of the ionic species at the surface. The ions can be concurrently quantified by recording the ion current at selected points along the collector array, for example, in one or more collector bins or compartments of the collector array, such that the total amount of material collected in each bin over a given time period can be determined. The measured ion currents can provide relative amounts of different components in the mixtures being analyzed and separated. Further, absolute amounts of a given component present can be determined with such measurement by employing mass-distinguishable internal standards.
In a preferred embodiment, a linear dispersion magnet is employed for mass separation of high velocity ionized components which avoids mass compression and potential loss of mass resolution at higher mass to charge ratios. Trapping can be performed using a linear collector array with equidistant bins. In another preferred embodiment, the soft-landed ions are neutralized by ion-pairing with counter-ions produced by electrolytic reduction of an auxiliary electrolyte to diminish side reactions and minimize or avoid chemical modification of trapped components.
The mass spectrometry-based method and instrument of this invention allows multichannel separation of ionized components of a mixture by mass, followed by non-destructive trapping of mass-separated ionized components, charge neutralization of ionized components and collection of separated mixture components.
In a specific embodiment, the invention provides a method of separation of mixture components comprising the steps of:
(a) nondestructive ionization of mixture components to generate ionized components;
(b) transmission of ionized components into a high vacuum region with simultaneous collisional cooling to near-thermal kinetic energies;
(c) acceleration of the ionized components having near-thermal kinetic energy to high kinetic energy (equal to or greater than about 1 keV) and refocusing of the accelerated ions to provide a focused ion beam for introduction into a kinetic energy analyzer;
(d) energy dispersion and spatial refocusing of the refocused accelerated ionized components as a function of their entrance trajectories and initial kinetic energy (i.e., kinetic energy after acceleration) to provide ionized components of selected kinetic energy for mass dispersion;
(e) mass dispersion and velocity refocusing of the ionized components of selected kinetic energy to generate mass separated ionized components;
(f) deceleration of the mass-separated ion components to a selected velocity and kinetic energy; and
(g) trapping of ion components separated by mass.
The method of this invention is carried out to obtain a desired amount of separated, trapped components of a mixture. The time that will be required to accumulate a desired amount of material is readily determined empirically for a given sample, the type and number of components in a sample and the amount of a given component in a sample that is to be collected. Non-destructive ionization can be carried out using any atmospheric pressure ionization method, but electrospray ionization (ESI) is particularly useful. ESI is typically carried out at ambient atmospheric pressure and the ionized components must then be transmitted to a region of high vacuum (about 10xe2x88x926 Torr) for mass separation. Mass dispersion of the accelerated, ionized components can be carried out in a magnetic field, preferably in a linear magnetic analyzer, resulting in linear mass dispersion of ions. The ions can be decelerated to a desired kinetic energy and in a preferred embodiment the ions are decelerated to a sufficiently low kinetic energy to minimize fragmentation on landing.
The method of the invention can be carried out in a mass spectrometer system comprising, in sequence along an ion""s trajectory through the system, an electrospray ion source, one or more ion guides, an ion acceleration lens, an electrostatic analyzer, a magnetic analyzer, a deceleration lens and a collector array with suitable ion transmission or transfer devices between device elements. The device elements other than the ion source are contained in a multi-chamber vacuum housing in which operating pressures are maintained by one or more pumping systems. The instrument employs appropriate differential pumping and conductance limits (determined by apertures size) between the chambers to achieve appropriate pressure levels in the different chambers.
As an alternative to an ESI source, an atmospheric pressure chemical ionization (APCI) source can be employed in the method of this invention. Atmospheric pressure chemical ionization is related to ESI and the ion source is similar to an ESI ion source. In addition to the electrohydrodynamic spraying process of ESI, a plasma is created by a corona-discharge needle at the end of the metal capillary. In this plasma, proton transfer reactions and some fragmentation can occur. Depending on the solvent used, only quasi molecular ions like [M+H]+, [M+Na]+ and M+ (in the case of aromatics), and/or fragments can be produced. Multiply charged molecules are typically not observed.
Both APCI and ESI provide molecular and quasi-molecular ions from which molecular weight information can be derived and as such are useful for the preparative method of this invention. ACPI is generally suitable for analyzing less polar compounds than ESI, but generally exhibits increased fragmentation compared to ESI. ACPI can provide coupling for samples at sampling flow rates up to about 1 ml/min.
Ionized components of the mixture are generated in the ion source at relatively high pressure, transmitted to a high-vacuum region, and accelerated by the acceleration lens to a selected high kinetic energy. Accelerated ions are passed through an electrostatic analyzer to disperse the ions by kinetic energy and focus them into a nearly paraxial beam (Mattauch-Herzog double focusing.) In the Mattauch-Herzog geometry, the electrostatic sector analyzer (ESA) refocuses the ions so that the velocity dispersion of the beam exiting the ESA can be compensated by the velocity dispersion of the magnet. Ions of paraxial trajectories are introduced into a magnetic analyzer which refocuses the ions by velocity and disperses the ions by mass, so that the ions can be trapped or collected as a function of mass. Ions are spatially separated by mass in the magnetic analyzer so that mass separation of components can be achieved by spatially-selective collection of mass-separated ions.
In an instrument of this invention, a collector array can be arranged to receive ions dispersed by mass by passage in the magnetic analyzer. Prior to collection the ions pass through a deceleration lens to decrease their velocity and kinetic energy to facilitate nondestructive landing on the collector array. The ions also pass through an optional, but preferred, deflector lens after exiting the magnet.
In a specific embodiment, the mass spectrometer-ion collection instrument of this invention comprises:
an atmospheric pressure chemical ionization (APCI) ionizer or an electrospray ionizer (ESI) for receiving a liquid sample containing one or more components and for generating one or more ionized components of the sample;
an ion transmission assembly for transmitting the one or more ion components generated by the electrospray ionizer into an acceleration lens;
an acceleration lens for imparting selected high kinetic energy to the one or more ionized components generated by the electrospray ionizer;
an electrostatic analyzer for dispersing and focusing the one or more accelerated ionized components as a function of kinetic energy and transmitting the one or more ionized components of selected kinetic energy;
a magnetic analyzer for receiving the one or more ionized components of selected high kinetic energy and for dispersing the ions received as a function of mass;
one or more electrostatic potential shielding devices (e.g., Faraday cages) between the acceleration lens and the electrostatic analyzer and between the electrostatic analyzer and the magnetic analyzer to transmit the one or more ionized components of selected high kinetic energy without loss or gain of kinetic energy;
an electrostatic deflector to simultaneously bend the trajectories of all mass-dispersed ions exiting the magnet analyzer and correct the trajectories for acceptance by the deceleration lens;
a deceleration lens intercepting the path of the one or more ionized components dispersed in the magnetic analyzer for decreasing the kinetic energy of the one or more mass-dispersed, ionized components; and
a collector array positioned to intercept the paths of the one or more decelerated, mass-dispersed ionized components from the magnetic array to spatially collect ions as a function of mass.
In a specific embodiment, the electrospray ionizer comprises a heated ion transfer interface with a counter flow of bath gas for receiving charged droplets and ionized components of sample generated in the electrospray ionizer and facilitating desolvation of the charged droplets to form ionized components of the sample. Further in a specific embodiment the ion transmission assembly comprises: a funnel lens for receiving ionized components from the ion transfer interface of the ionizer and an octopole ion guide for receiving ionized components exiting the funnel lens and transporting the ions to the acceleration lens. On passage through the octopole ion guide, ions undergo collisions with residual background gas for translational cooling. Extraction ion optics between the octopole and the acceleration lens comprise an extraction lens for extracting ions from the octopole and an Einzel lens for refocusing the ions into the acceleration lens.
In a specific embodiment, the vacuum housing of the instrument comprises four compartments or chambers which are held at different pressures and are separated by low conductance apertures. The ion transfer interface and funnel lens are held at a pressure of about 0.1 to 5 Torr (Chamber 1). The octopole ion guide is held at about 5xc3x9710xe2x88x924 to 1xc3x9710xe2x88x922 Torr (Chamber 2). The acceleration lens and electrostatic analyzer are held at a pressure of about 1-5xc3x9710xe2x88x926 Torr (Chamber 3). The collector, deflector and deceleration lens are housed in Chamber 4 which is connected to the magnetic field tube and held at a pressure of 1xc3x9710xe2x88x926 to 1xc3x9710xe2x88x925 Torr.
In a preferred embodiment, the magnetic analyzer is a linear magnetic analyzer as described in U.S. Pat. No. 6,182,831 (issued Feb. 6, 2001) which is incorporated by reference herein in its entirety for the description of a linear magnetic analyzer.
The specific combination of device elements in the specific embodiments herein facilitate efficient separation and rapid collection of components of sample mixtures.
The invention also relates to a method for collecting ionic species at rates sufficiently high for practical application. The instrument of this invention can be employed, for example to generate and collect ionic species at rates of 10 picomoles/h or higher. More specifically, the invention provides a method for collecting or landing of ionic species into a matrix or onto a substrate at the collector. Ions can be soft landed without significant fragmentation or rearrangement. Alternatively, ions can be landed with a selected, controlled kinetic energy allowing ion rearrangement, ion fragmentation and/or ion reaction. In another alternative, the ions and the matrix or substrate can be selected such that the matrix or substrate is modified, either functionally, chemically or structurally by the ions captured. Of particular interest are matrices of inorganic or organic polymers which can be modified by reaction or interaction with one or more ions.